Straightforward Entry to S -Glycosylated Fmoc-Amino Acids and Their Application to Solid Phase Synthesis of Glycopeptides and Glycopeptidomimetics

Article abstract of DOI:10.1021/ol503664t

Streamlined access to S-glycosylated Fmoc-amino acids was developed. The process provides diverse glycosylated modified amino acids in high yield and stereoselectivity taking advantage of the in situ generation of a glycosylthiolate obtained from carbohyd

Full text of DOI:10.1021/ol503664t

Letter
pubs.acs.org/OrgLett
Straightforward Entry to S‑Glycosylated Fmoc-Amino Acids and Their
Application to Solid Phase Synthesis of Glycopeptides and
Glycopeptidomimetics
Daniela Comegna,† Ivan de Paola,† Michele Saviano,‡ Annarita Del Gatto,† and Laura Zaccaro*,†
†Institute of Biostructures and Bioimaging, National Research Council, Napoli, Italy
‡Institute of Crystallography, National Research Council, Bari, Italy
S
* Supporting Information
ABSTRACT: Streamlined access to S-glycosylated Fmoc-
amino acids was developed. The process provides diverse
glycosylated modified amino acids in high yield and stereo-
selectivity taking advantage of the in situ generation of a
glycosylthiolate obtained from carbohydrate acetates in a few
steps. Mild basic conditions make the conjugation reaction
compatible with Fmoc-iodo-amino acids. To validate the
strategy the glycosylated building blocks were used for SPPS
and the unprecedented incorporation of a long thio-
oligosaccharide to the peptide chain was demonstrated.
rug discovery based on peptides and proteins is a widely
explored area in biomedical research that very often relies
the corresponding glycosyl bromides. Access to these 1-thiosugar
derivatives is a tedious, time-consuming process and requires
harsh acidic conditions for the generation of the 1-bromo
intermediate, which can be an especially demanding issue when
applying the method to longer oligosaccharides. In fact, a
postsynthetic strategy of trisaccharide S-conjugation with a
peptide was already described,12 but saccharide sequences longer
than two residues were never thio-anchored to Fmoc-amino
acids prior to their solid-phase incorporation into a growing
chain.
On this basis, we directed our effort toward the implementa-
tion of an operationally simple synthetic approach to S-
glycosylated Fmoc-protected amino acids, which is endowed
with both the compatible direct use of commercial Fmoc-amino
acids and the applicability to carbohydrates more complex than
mono- or disaccharides. To this aim, we have suitably adapted a
straightforward strategy recently described for the synthesis of
thioglycosides, entailing the conversion of per-O-acetylated
carbohydrates into a glycosyl thiolate intermediate which is
entrapped in situ with a suitable electrophile.13 The synthetic
sequence is advantageous because the sequential generation of
the two requisite intermediates (namely, a glycosyl iodide and a
glycosylthiouronium) takes very short reactions, and no
purification of any saccharide intermediate is needed.
D
on the “peptidomimetic approach”. As a matter of fact,
introduction of modified amino acids or higher homologues
thereof results in an expanded molecular diversity that can be
exploited as a very effective tool in medicinal chemistry.1 Indeed,
peptidomimetics based on β- and γ-amino acids feature
enhanced in vivo stability, but can also be of valuable interest
for the investigation of new highly ordered architectures.2,3
Over the past years, the critical role played by the protein
glycosylation in biology has been widely unveiled, as witnessed
by the involvement of glycoproteins in a wide set of events such
as cell adhesion and proliferation, trafficking, cell−cell
recognition, inflammation, virulence and host immune re-
sponse.4 Glycosylation also plays a pivotal role in protein folding
and proteolytic stability, and it is widely investigated for tuning
the biological activity of non-naturally glycosylated peptides and
proteins.5
In this regard, synthetic access to homogeneous natural or
modified glycosylated peptides is a relevant topic in organic
chemistry and different synthetic conjugation methods have
been developed,6 providing naturally occurring O- and N-linked,
and uncommon C- and S-linked, glycopeptides. In particular, the
thioglycoside bond is well-known to be chemically and
enzymatically more stable than the O- linked counterpart, and
it is well tolerated in biological systems because of its isosteric
mimicry.7 To date, several methods for the assembly of S-
glycosylated peptides have been reported,8 but very few of them
are compatible with use of Fmoc-protected amino acids.9 As to
the saccharide moiety of the reported glycosylated Fmoc-amino
acids and peptides, the known synthetic strategies entail
preliminary generation and purification of a glycosylthiol,9b,c,10
a glycosylthioacetate,9a or a glycosylthiomethylating agent11 via
First, we have obtained iodo β- and γ-amino acids starting from
Fmoc-L-aspartic and Fmoc-L-glutammic acids 4- and 5-tert-butyl
esters respectively (a−b, Scheme 1) and iodo α-amino acids
starting from corresponding 1-tert-butyl esters (c−d, Scheme 1),
one of which belongs to the D-series (e, Scheme 1).9c The free
Received: December 19, 2014
© XXXX American Chemical Society
A
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Letter
a
Scheme 1. Preparation of Iodo-Amino Acids
Table 1. S-Glycoconjugation Reactions of Various Sugar
Acetates with Iodo-Amino Acids
a
product and yield (%)
entry
1
sugar
β- and γ-iodo aa
α-iodo aa
D-glucose (1)
1a (60 β only)
1b (66 β only)
1c (82 β only)
1d (88 β only)
1e (80 β only)
2c (90 α only)
2d (83 α only)
3c (90 β only)
3d (86 β only)
4c (78 β only)
4d (82 β only)
5c (60 β only)
6c (60 β only)
6d (62 β only)
7c (83 β only)
2
3
4
D-mannose (2)
D-galactose (3)
L-fucose (4)
2a (65 α only)
2b (68 α only)
3a (70 β only)
3b (74 β only)
4a (60 β only)
4b (58 β only)
bc
,
a
5
6
glucosamine (5)
lactose (6)
Reagents and conditions: (i) Isobuthylchloroformate, NMM, THF, 0
°C, 1 h; (ii) NaBH4, THF/H2O, 0 °C, 20 min; (iii) I2, PPh3, imidazole,
THF, rt, 1 h.
6a (53 β only)
6b (55 β only)
7b (69 β only)
8b (46 β only)
c
7
8
maltotriose (7)
c
maltotetraose (8)
acidic functions were reduced to the corresponding alcohols and
then converted into iodides14 (Scheme 1).
a
b
Isolated overall yields. See Supporting Information for experimental
c
details. The reactions reported are the only ones carried out.
The saccharide moiety was instead suitably elaborated through
the very fast three-step procedure shown in Scheme 2. In the first
step, the per-O-acetylated sugar precursor was treated with
iodine and triethylsilane in dichloromethane under reflux to
afford the corresponding anomeric iodide15 in quantitative yield
and within a few minutes. After a simple extractive workup, the
crude glycosyl iodide was quickly converted into the
corresponding isothiouronium salt by treatment with thiourea
in acetonitrile at 60 °C; to the same vessel, the Fmoc-protected
amino acid derivative was then added along with triethylamine to
perform the S-conjugation in a one-pot fashion through a
nucleophilic attack of the sugar thiolate anion to an Fmoc-iodo-
amino acid, a process occurring with both high anomeric
selectivity and yield (Table 1) and without the generation of
disulfide side products.16
Optimization of the results required a thorough screening of
conditions for the critical last step of the sequence by examining
several parameters such as the solvent, sugar-to-amino acid
stoichiometric ratio, temperature, and amount of the base. Not
unexpectedly, the amount of TEA was the most critical
parameter to affect the yield; when a large excess of the base
(4−12 equiv) was used, the conjugation yield was drastically
reduced by the Fmoc cleavage and subsequent generation of the
adduct between the thiolate and dibenzofulvene arising from the
Fmoc release. On the other hand, use of a moderate excess of the
base (2 equiv) proved sufficient for promoting the desired
coupling process without any loss of the Fmoc protecting group.
The optimization study also indicated acetonitrile as a good
solvent for the final step and that the glycoconjugation is fast
enough to proceed at room temperature within 3−4 h.
Remarkably, the overall conversion of the per-O-acetylated
precursor to the S-glycosylated Fmoc-amino acid did not exceed
5 h and required a single purification step by column
chromatography. In addition, all reactions of the sequence in
Scheme 2 were performed under air without any special
experimental precaution. The wide versatility of the proposed
strategy was demonstrated by both the successful application to a
wide range of mono- (1−5) and disaccharides (6) and the
unprecedented glycoconjugation of Fmoc-amino acids with
longer sequences such as the maltotriose trisaccharide 7 and
maltotetraose tetrasaccharide 8 (Figure 1). Only for N-acetyl
glucosamine, the relatively stable anomeric chloride 517 was used
instead of the corresponding iodide to react with thiourea in
refluxing acetone to give the thiouronium salt.16 In this case, the
best yield was achieved by exchanging acetone for acetonitrile
prior to performing the S-alkylation step. As proof of concept, the
conjugation reactions for sugars 5, 7, and 8 have been carried out
only with iodo-amino acids b and c.
a
Scheme 2. Synthetic Sequence for S-Glycosylated Amino Acids Formation
a
(i) I2, Et3SiH, CH2Cl2, reflux, 5 min; (ii) thiourea, CH3CN, 60 °C, 60 min; (iii) 0.7 equiv of a−e, 1.5 equiv of Et3N, rt, 3−4 h. Yields reported
below in Table 1.
B
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a
Scheme 3. Unmasking of Carboxyl Group
a
Reagents and conditions: (i) CH2Cl2/TFA 4:1, 0 °C, 2 h.
a
Scheme 4. Glycopeptides by SPPS and Deacetylation
Figure 1. Starting peracetylated carbohydrates.
All the S-glycosyl amino acids were obtained with high 1,2-
trans stereoselectivity; namely, pure β-anomers were obtained
from gluco (1a−e, 5d, 7b−c, and 8b) or galacto-configured sugars
(3a−d, 4a−d), whereas pure α-anomers were obtained from a
manno-precursor (2a−d).
It is worth noting that the nucleophilic substitution reactions
to more accessible electrophilic centers of iodo α-amino acids
(c−e) are significantly more high-yielding (60−90%) than in the
case of β- (a) and γ- (b) amino acid halides (46−74%) bearing
the electrophilic carbon much closer to the bulky 9-fluorenyl-
methoxycarbonyl group.
It should be outlined that acetyl protecting groups play a
beneficial role at multiple stages of the synthesis. First, they
guarantee the above-mentioned 1,2-trans selectivity due to their
possible participation effect serving in the generation of the
thiouronium intermediate; in addition, their electron-with-
drawing nature allows the oligosaccharide chains to be unaltered
by the acidic conditions needed both in the early stage of
anomeric iodination of the reducing terminus and in all the acidic
conditions to which the S-glycosyl Fmoc-amino acids are
exposed prior to and after its incorporation into the peptide
sequence. Last, their presence ensures the protection of hydroxyl
groups toward esterification when a high concentration of amino
acid was used during the peptide elongation.18
To validate the scope of our strategy, some of the obtained
building blocks were employed in Fmoc solid-phase peptide
synthesis. For this purpose, amino acids 1c, 1a, and 8b were
treated with TFA in dichloromethane to hydrolyze the tert-butyl
ester function affording the final Fmoc-protected amino acids 1h,
1f, and 8g (Scheme 3) in good yield (see Supporting
Information). We designed our glycopeptides on an enkephalin
analogue,19 a pharmacophore contained in many opioids,20
which possesses an improved analgesic effect due to a more
efficient uptake through the blood−brain barrier arising from a
simple glucosylation of a serine residue (9, Scheme 4).21 Inspired
by this model, we synthesized three S-glycosylated modified
peptides highlighting the versatility of the strategy through the
feasible introduction of glycosylated α-, β-, and γ-amino acids
(10−12, Scheme 4). The glycoamino acid building blocks so
a
Reagents and conditions: (i) MeOH/NH4OH 4:1, rt, overnight.
prepared were in turn incorporated into the peptide sequence by
a standard SPPS technique.
Notably, the solid-phase synthesis was found effective even
with amino acids bearing a tetrasaccharide moiety and was also
compatible with the assembly of glycopeptides with different
sugars appended to the same peptide backbone.
In conclusion, we have developed a new general strategy for
the linear assembly of S-glycopeptides and S-glycopeptidomi-
metics by standard Fmoc solid-phase synthesis. Upon incorpo-
ration of glycosylated α-amino acids, the pendant sugar moiety is
linked to a homocysteine or homocysteine-like residue, whereas
in the case of the incorporation of the corresponding β- and γ-
glycoamino acids the sugar moiety looks linked to a cysteine side
chain.
The method relies on a very fast and operationally simple
synthesis of Fmoc-protected S-linked glycosyl amino acid
building blocks starting from commercially available per-O-
acetylated sugars and Fmoc/tert-butyl ester protected amino
acids, both converted into the corresponding iodides. In a one-pot
C
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Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(11) Zhu, X.; Pachamuthu, K.; Schmidt, R. R. Org. Lett. 2004, 6, 1083−
fashion the glycosyl iodide was turned into the anomeric thiolate
via a thiouronium intermediate and then coupled with the iodo-
amino acid to quickly afford the conjugated product with high
stereoselectivity and yield. The Fmoc-protective group was
found to be compatible with the optimized reaction conditions,
and no protecting-group manipulation is needed except for the
unmasking of the carboxylic function. Furthermore, the entire
process requires just one purification step and short experimental
times in comparison with other methods. The strategy proved to
be applicable to a wide range of carbohydrates namely mono-, di-,
and oligosaccharides, and it could be a useful tool in biomedical
and medicinal chemistry for the streamlined construction of new
glycopeptide targets of biological interest.
1085.
(12) Galonic, D. P.; van der Donk, W. A.; Gin, D. Y. Chem.Eur. J.
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2007, 72, 6097−6106. On other kinds of aglycons: (b) Comegna, D.;
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Bedini, E.; Parrilli, M. Tetrahedron 2008, 64, 3381−3391. (d) Bedini, E.;
Forte, G.; De Castro, C.; Parrilli, M.; Dalla Cort, A. J. Org. Chem. 2013,
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(14) Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G.
Tetrahedron 1995, 51, 12337−12350.
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(15) (a) Adinolfi, M.; Iadonisi, A.; Ravida, A.; Schiattarella, M.
Tetrahedron Lett. 2003, 44, 7863−7866. (b) Giordano, M.; Iadonisi, A.
Eur. J. Org. Chem. 2013, 125−131.
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ASSOCIATED CONTENT
■
S
* Supporting Information
(17) RajanBabu, T. V.; Ayers, T. A.; Halliday, G. A.; You, K. K.;
Calabrese, J. C. J. Org. Chem. 1997, 62, 6012−6028.
Full experimental details, characterization of all compounds, 1H
and 13C NMR spectra of sugar conjugates, NMR spectra, and
LC-MS profiles of glycopeptides are reported. This material is
available free of charge via the Internet at http://pubs.acs.org.
(18) It is interesting to note that in a recent report describing the
possible direct attachment of unprotected mono- and disaccharides to a
resin bound growing peptide, the resulting glycosylated intermediate
was per-O-acetylated just after the glycosylation step and prior to
performing the elongation of the peptide chain through standard solid-
phase protocols: Novoa, A.; Barluenga, S.; Serbaab, C.; Winssinger, W.
Chem. Commun. 2013, 49, 7608−7610.
AUTHOR INFORMATION
■
Corresponding Author
(19) Yamamoto, N.; Takayanagi, A.; Yoshino, A.; Sakakibara, T.;
Kajihara, Y. Chem.Eur. J. 2007, 13, 613−625.
*E-mail: lzaccaro@unina.it.
Notes
(20) Michell, S. A.; Pratt, M. R.; Hruby, V. J.; Polt, R. J. Org. Chem.
2001, 66, 2327−2342.
The authors declare no competing financial interest.
(21) Strand, F. L. Endorphins, Chemistry, Physiology, Pharmacology and
Clinical Relevance; Marcel Dekker: New York, NY, 1982.
(22) Egleton, R. D.; Michell, S. A.; Huber, J. D.; Janders, J.; Stropova,
D.; Polt, R.; Yamamura, H. I.; Hruby, V. J.; Davis, T. P. Brain Res. 2000,
881, 37−46.
ACKNOWLEDGMENTS
■
We are grateful to Mr. Leopoldo Zona (National Research
Council, Naples) for NMR technical assistance and Mr. Luca De
Luca (National Research Council, Naples) for computer
assistance. This study has been supported by the Italian Ministry
of University and Research (PRIN2010-2011-prot.
2010C4R8M8_007).
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